Cyclin dependent kinase (CDK)4 inhibitors and their use for treating cancer

ABSTRACT

Certain derivatives of acridones and benzothiadiazines have been found to have anti-cancer properties by virtue of their specific inhibition of the cyclin D dependant kinase CDK4. These molecules inhibit CDK4 activity more than they inhibit the activity of other such kinases (e.g. CDC2 and CDK2). This specificity results in an improved therapeutic index when used as drugs to treat susceptible cancers.

This application claims the benefit of provisional application No.60/044,256 filed Apr. 28, 1997.

FIELD OF THE INVENTION

The present invention concerns compounds that inhibit cyclin-dependentkinases, particularly the cyclin-dependent kinase CDK4, and methods fortreating cancers using such compounds.

BACKGROUND OF THE INVENTION

Physiology

In a normal cell CDK4:cyclin D kinase holoenzyme phosphorylates theretinoblastoma protein (Rb) to form hyperphosphorylatedretinoblastoma-phosphate (Rb-p). The hyperphosphorylation ofretinoblastoma protein results in the release of Rb-p associatedtranscription factors that allow cell cycle progression beyond the G1checkpoint, thereby promoting cell proliferation (Schrr et al., U.S.Pat. No. 5,723,313, (1998)).

The p16 gene (also known as CDKN2, MST1, and CDK4I) encodes the proteinp16^(INK4A), which inhibits the cyclin-dependent kinase (CDK)4:cyclin Dcomplex (Serrano, et al., Nature 366: 704-7 (1993)). Defects in thep16/CDK4:cyclinD/Rb pathway may lead to tumor formation. Geneticalteration or over expression of CDK4 and CyclinD1 has been observed invarious tumor cell types. In addition, alterations of p16 have beendescribed in various histologic types of human cancers includingretinoblastoma, astrocytoma, melanoma, leukemia, breast cancer, head andneck squamous cell carcinoma, malignant mesothelioma, and lung cancer(Kamb et al., Science 264: 436-40 (1994); Noborie et al., Nature 368:753-56 (1994); Walker et al., Cancer Res. 55: 20-3 (1995) and Nakagawaet al., Oncogene 11: 1843-51 (1995)).

Acridones and Benzothiadiazines

Acridones and benzothiadiazines (BTDs) are classes of known cyclic arylcompounds. Certain known acridones or BTDs have pharmacological effects.For example, BTDs have been investigated as diuretics (See de Tullio etal., J. Med. Chem). Fajans and Floyd (Ann. Rev. Med. 30:313-329, 1982)disclose the use of “diuretic benzothiadiazine, e.g. trichlormethiazide”as a hyperglycemic in the treatment of insulinomas. Fajans and Floyd,however, do not teach the use of BTDs to affect cancers directly. Theprior art, as understood, does not appear to teach the use of BTDs fortheir direct antineoplastic effect in the specific inhibition of CDK4dependent tumors.

Particular acridones and acridines are known. For example,(C₁₈H₁₉N₃O₂—HCl) has been mentioned in a paper concerned with theanti-tumor activity of linear tri-cyclic carboxamides (Palmer et al., J.Med. Chem (US) 31 (4) pgs.707-721, 1988). Interestingly, the Palmer etal. paper states that this compound is “inactive” (page 711, column 1,paragraph 3).

The basic thioacridone ring structure was described in DeLeenheer etal., J. Pharm. Sci. 60:1238-1239, 1971, and is shown below.

1-nitro-9-acridone, 1-nitro-10-(3-N,N-dimethylaminopropryl)-9-acridone,1-amino-2,4-diethylthio-9-acridone and a number of acridine derivativeshave been disclosed by Weltrowski et al. (Pol. J. Chem Technol.56:77-82, 1982). This paper, however, deals exclusively with thesynthesis of nitroacridines and does not discuss any biological activityor mechanism of biological action. But, the title of the Weltrowskiarticle refers to tumor inhibition, and the footnote states that thework was supported by the Polish National Cancer Program.

SUMMARY OF THE INVENTION

The present invention concerns acridones, benzothiadiazines andderivatives thereof that are useful for treating cancers. The inventionalso concerns methods for using these compounds as CDK4 inhibitors totreat cancers.

There are a number of dreadful and relatively common cancers that havebeen shown to involve alterations in p16. These cancers include lungcancer, breast cancer, melanoma, leukemia, retinoblastoma, astrocytoma,head and neck squamous cell carcinoma and malignant mesothelioma.Expression of normal p16 protein in tumor cells with alterations of p16results in restoration of cell-cycle regulation, decreased cell growthand decreased tumorigenicity in vivo. Because the only known function ofp16 is inhibition of CDK4 kinase activity, cancers with alterations ofp16, including those listed above, are likely to be sensitive to CDK4inhibitors. Prior inhibitors of cyclin-dependent kinases, such asflavopiridole, staurosporin, and UCN-01, inhibit CDC2 and CDK2 as wellas the intended target, CDK4. This lack of specificity producespathological side effects, such as bone marrow and gastrointestinaltoxicities, and limits their clinical application.

As a result, there is a need for drugs for treating CDK4 sensitiveneoplasms that minimize toxic side effects caused by concomitantinhibition of CDC2 and CDK2. The compounds claimed in this applicationinhibit CDK4 to a far greater extent than CDC2 or CDK2 and thereforesatisfy this need.

One example of a novel compound of the present invention is3-amino-9-thio(10H)-acridone. This compound and others can be used toform therapeutic compositions. One embodiment of such a compositioncomprises a therapeutically effective amount of a compound selected fromthe group consisting of a benzothiadiazine, a thioacridone, or mixturesthereof. The compound has an IC₅₀ for CDK4 of less than about 10 μM,preferably from about 1 μM to about 7 μM, an IC₅₀ for CDC2 of greaterthan about 60 μM, preferably greater than about 100 μM, an IC₅₀ forCDK2/A of greater than about 100 μM, an IC₅₀ for CDK2/E of greater thanabout 80 μM, and preferably greater than about 100 μM.

The specificity of the compounds for inhibiting CDK4 can be expressed asa ratio of the IC₅₀ values for other enzymes relative to CDK4. Suchcompositions typically comprise a compound selected from the groupconsisting of a benzothiadiazine, a thioacridone, or mixtures thereof,the compound having an IC₅₀ ratio for CDC2:CDK4 of greater than about8.5, typcially greater than about 20, preferably greater than about 60;an IC₅₀ ratio for CDK2/A:CDK4 of greater than about 14, typicallygreater than about 20, and preferably greater than about 60; and an IC₅₀ratio for CDC2/E:CDK4 of greater than about 11.5, typically greater thanabout 20, and preferably greater than about 60.

The invention also provides a composition comprising an effective amountof a compound according to Formula 1

where m is 0 or 1, n=m, R₁—R₄ are independently selected from the groupconsisting of H, —NH₂ and lower alkoxy, where with m=1 one of R₁—R₄ isan amine bonded to R′ to form an arylamide, or Formula 2

where R and R₁ are independently carbon or nitrogen, where if R₁=carbonX is hydrogen, halogen, aryl or alkoxy, and R₂ is selected from thegroup consisting of lower alkyl and aryl amino. The composition also cancomprise mixtures of compounds satisfying Formula 1 and/or Formula 2.The composition can further include, without limitation, additivesselected from the group consisting of carriers, diluents, excipients,diagnostics, direct compression buffers, buffers, stabilizers, fillers,disintegrates, flavors, colors, and mixtures thereof.

A method for inhibiting the growth of living cells also is described.The method comprises providing a compound selected from the groupconsisting of a benzothiadiazine, a thioacridone, or mixtures thereof,as described above. An effective amount of the compound, a mixture ofcompounds, or a composition comprising the compound or mixture ofcompounds, is administered to a subject to inhibit the growth of livingcells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(A)-1(I) are dose-response curves showing the effect of Compound5 on various cancer cell lines in culture.

FIGS. 2(A)-2(C) shows mean plots of data from FIGS. 1A-1I, wherein theleft-hand mean plot is of GI₅₀ data, the middle mean plot is of TGIdata, and the right-hand mean plot is of LC₅₀ data.

FIGS. 3(A)-3(I) are dose-response curves showing the effect of Compound7 on various cancer cell lines in culture.

FIGS. 4(A)-4(C) shows mean plots of data from FIGS. 3A-3I, wherein theleft-hand mean plot is of GI₅₀ data, the middle mean plot is of TGIdata, and the right-hand mean plot is of LC₅₀ data.

FIGS. 5(A)-5(I) are dose-response curves showing the effect of Compound8 on various cancer cell lines in culture.

FIGS. 6(A)-6(C) shows mean plots of data from FIGS. 5A-5I, wherein theleft-hand mean plot is of GI₅₀ data, the middle mean plot is of TGIdata, and the right-hand mean plot is of LC₅₀ data.

FIGS. 7(A)-7(I) are dose-response curves showing the effect of Compound4 on various cancer cell lines in culture.

FIGS. 8(A)-8(C) shows mean plots of data from FIGS. 7A-7I, wherein theleft-hand mean plot is of GI₅₀ data, the middle mean plot is of TGIdata, and the right-hand mean plot is of LC₅₀ data.

FIGS. 9(A)-9(I) are dose-response curves showing the effect of Compound6 on various cancer cell lines in culture.

FIGS. 10(A)-10(C) shows mean plots of data from FIGS. 9A-9I, wherein theleft-hand mean plot is of GI₅₀ data, the middle mean plot is of TGIdata, and the right-hand mean plot is of LC₅₀ data.

FIGS. 11(A)-11(I) are dose-response curves showing the effect ofCompound 3 on various cancer cell lines in culture.

FIGS. 12(A)-12(C) shows mean plots of data from FIGS. 11A-11I, whereinthe left-hand mean plot is of GI₅₀ data, the middle mean plot is of TGIdata, and the right-hand mean plot is of LC₅₀ data.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

DEFINITIONS

Particular terms and phrases used herein typically have the meanings setforth below. These definitions are provided solely for convenience andshould not be interpreted to limit the invention to a scope less thanthat known to a person of ordinary skill in the art.

“3-ATA” means 3-amino-9-thio(10H)-acridone.

“BTD” means benzothiadiazine.

“Neoplasm” and “cancer” both refer to any cell or tissue wherein growthand cell division have become uncoupled from the normal regulatoryconstraints of the cell cycle to produce a pathological state.

“Tumor” is any neoplasm and includes both solid and non-solid neoplasms.

“Inhibitory concentration” or “IC₅₀” means the drug concentration at 50%inhibition of kinase activity (μM).

“Therapeutically effective anti-neoplastic amount” means an amountsufficient to prevent advancement, or to cause regression of, aneoplasm.

“CDK4” and “CDK4/A” refer to the CDK4:cyclin D1 kinase holoenzyme.

“CDK4 inhibitor” refers to compounds that inhibit the kinase activity ofCDK4.

“CDK4 inhibition” refers to inhibition of the kinase activity of CDK4.

“CDK2”, when used alone, refers to both CDK2:Cyclin A and to CDK2:CyclinE

“CDC2” and “CDC2/A” refer to CDC2:Cyclin A holoenzyme.

“CDK2/A” refers to CDK:Cyclin A holoenzyme.

“CDK2/E” refers to CDK2:Cyclin E holoenzyme.

“Cancers specifically inhibited by CDK4 inhibitors” means allneoplastically transformed cells and tissues, the growth and/or cellcycle of which is affected by a CDK4 inhibitor.

A cell “susceptible to CDK4 inhibitors” or “susceptible to CDK4inhibition” is a cell for which CDK4 inhibitors alter growth or cellcycle.

“Specific inhibition” or “specific inhibitory activity” of the compoundsof the invention means that the compounds inhibit CDK4 to a greaterextent than they inhibit CDC2 or CDK2.

“Lower-alkyl” means a single-bonded branched or unbranched hydrocarbonchain having from about one to about ten carbon atoms, including allposition and stereoisomers.

Compounds

Compounds of the present invention satisfy either Formula 1(acridone-like structures) or Formula 2 (benzothiadiazine-likestructures) below.

With reference to Formula 1, m is 0 or 1, and n=m. R₁-R₄ areindependently selected from the group consisting of H, —NH₂ and loweralkoxy. With m=1, at least one of R₁-R₄ is an amine and R′ is bonded tothe amine to form an arylamide.

With reference to Formula 2, R and R₁ are independently carbon ornitrogen. If R₁=carbon X is hydrogen or halogen. R₂ is selected from thegroup consisting of lower alkyl and aryl amino.

Compounds according to both Formula 1 and 2 show specific inhibitoryactivity against CDK4. This inhibition may be due to inhibition offormation of the CDK4:cyclinD kinase holoenzyme or to competitivebinding of the inhibitor with the kinase substrate or to ATP-dependentcompetitive effects or some other interaction.

Structural formulas for particular compounds of the invention areprovided below as Compounds 1-6.

3-Amino-10H-acridine-9-thione

1,4-Dimethoxy-10H-acridine-9-thione

2,2′-Biphenyldiamine,bis[N,N′-[3-(amidonmethylamino)-10H-acridine-9-thione]]

4-(4-Fluorobenzylamino)-1,2,3-benzothiadiazine-1,1-dioxide

3-Chloro-4-methyl-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide

3-Chloro-4-ethyl-4H-benzo[e][1,2,4]thiadiazine 1,1-dioxide

Synthesis of Compounds

The compounds of the invention were obtained from and are maintained atthe Drug Synthesis and Chemistry Branch, National Cancer Institute.Syntheses of related compounds are known in the literature. For example,the following references described the syntheses of certain relatedcompounds: Pascal de Tullio et al., “3- and 4-Substituted4H-Pyrido[4,3-e]-1,2,4-thiadiazine 1,1-Dioxides as Potassium ChannelOpeners: Synthesis, Pharmacological Evaluation, and Structure—ActivityRelationships,” J. Med. Chem., Vol. 39, pp. 937-948 (1996); Bernard A.Dumaitre et al., U.S. Pat. No. 5,604,237; Hamprecht et al., U.S. Pat.No. 4,075,004; Magatti U.S. Pat. No. 4,468,396; Brian D. Palmer et al.,“Potential Antitumor Agents. 54. Chromophore Requirements for in VivoAntitumor Activity Among the General Class of Linear TricyclicCarboxamides,” J. Med. Chem., Vol. 31, pp. 707-712 (1988); N. Dodic etal., “Synthesis and Activity Against Multidrug Resistance in ChineseHamster Ovary Cells of New Acridone-4-Carboxamides,” J. Med. Chem., Vol.38, pp. 2418-2426 (1995); Marek Welt4rowski et al., “Research on TumourInhibiting Compounds, Part LXX, Reactions of 1-Nitroacridines withEthanethiol,” Polish Journal of Chemistry, pp. 77-82 (1982).

Compositions

Compounds satisfying either Formula 1 or 2 above may be formulated aspharmacological compositions containing a therapeutically effectiveanti-neoplastic amount of the compound(s). Such compositions may furthercomprise, without limitation, inert carriers, diluents, excipients,diagnostics, direct compression buffers, buffers, stabilizers, fillers,disintegrates, flavors, colors, other materials conventionally used inthe formulation of pharmacological compositions and mixtures thereof.

Method

The method of the present invention comprises administering to a subjecta therapeutically effective anti-neoplastic amount of a compound,mixture of compounds, or composition or compositions comprising thecompound or compounds; to effect a change in the physiology of aneoplasm. One of ordinary skill in the art will realize that thetherapeutically effective anti-neoplastic amount may vary. Anti-tumoragents generally are dosed as mass-per-unit-body surface area of thesubject. It currently is believed that a therapeutically effectiveanti-neoplastic amount of the disclosed compounds may be from about 1 μgto about 10 g per m² of body surface area, more preferably from about 1mg to about 900 mg per m² of body surface area. Moreover, it typicallyis desirable to provide as large a dose as a subject will tolerate.

The compound(s) or compositions may be administered by any number ofmethods including, but not limited to, intravenously, topically, orally,intramuscularly, subcutaneously, intraperitoneally. Currently,intravenous and oral administration are considered the preferable routesof administration.

Biological Methods and Results

Tables 1 and 2 provide IC₅₀ data for compounds representative of thepresent invention. These tables demonstrate that the IC₅₀ value ofcompounds according to the present invention for CDK4 generally is lessthan about 10 μM, and preferably is less than about 7 μM. The bestcompound, solely in terms of its IC₅₀ value for CDK4, is compound 5 withan IC₅₀ of 1.1 μM. But, compounds 7 and 8 also have IC₅₀ values of lessthan 2 μM, namely 1.4 μM and 1.7 μM respectively.

The compounds of the present invention also are quite specific forinhibition of CDK4. This is reflected in the IC₅₀ ratios reported inTables 1 and 2, with the IC₅₀ for CDK4 being the denominator in theratio e.g., (IC₅₀ CDC2)/(IC₅₀ CDK4). Thus, the lower the IC₅₀ is forCDK4 and the higher it is for the other complexes, the more specific thecompound is for CDK4.

The CDC2/A:CDC4 ratios in Tables 1 and 2 range from about 8 to greaterthan 72. The best compound with respect to specificity between CDK4 andCDC2 is compound 7, with an IC₅₀ for CDK4 of 1.4 μM, an IC₅₀ for CDC2of >100 μM, and an (IC₅₀ CDC2):(IC₅₀ CDK4) of >71.5.

Compound 3 (3-ATA) has an IC₅₀ for CDK4 of 6.8 μM, an IC₅₀ for CDC2 of60 μM, and an (IC₅₀ CDC2):(IC₅₀ CDK4) of 8.8.

Compound 4 has an IC₅₀ for CDK4 of 2.2 μM, an IC₅₀ for CDC2 of >100 μM,and an (IC₅₀ CDC2):(IC₅₀ CDK4) of >45.

Compound 5 has an IC₅₀ for CDK4 of 1.1 μM, an IC₅₀ for CDC2 of >70 μM,and an (IC₅₀ CDC2):(IC₅₀ CDK4) of >63.6.

Compound 6 has an IC₅₀ for CDK4 of 5.0 μM, an IC₅₀ for CDC2 of >100 μM,and an (IC₅₀ CDC2):(IC₅₀ CDK4) of >71.5.

Compound 8 has an IC₅₀ for CDK4 of 1.7 μM, an IC₅₀ for CDC2 of >100 μM,and an (IC₅₀ CDC2):(IC₅₀ CDK4) of >58.8.

IC₅₀ and IC₅₀ ratio data for other kinases are summarized in Tables 1and 2 below.

Compounds satisfying Formulas 1 and 2 have been subjected to biologicalassays to determine inhibition of the cyclin dependent kinases CDK4,CDC2, CDK2/A and CDK2/E. The experimental procedures for thesebiological methods and assays are provided below in the Examples.Results of these assays for representative compounds are provided belowin Tables 1 and 2.

TABLE 1 IC₅₀ value (μM) Formula Ratio Ratio Ratio Name CDK4/D1 CDC2/ACDC2A:CDK4 CDK2/A CDK2/A:CDK4 CDK2/E CDK2/E:CDK4 Compounds structurallyrelated to 3-ATA Formula 6.8 60 8.8 >100 >14.7 80 11.8 3 Formula2.2 >100 >45 >100 >45 >100 >45 4 Formula 1.1 70 63.6 >100 >91 >100 >91 5

TABLE 2 IC₅₀ value (μM) Formula Ratio Ratio Ratio Name CDK4/D1 CDC2/ACDC2A:CDK4 CDK2/A CDK2/A:CDK4 CDK2/E CDK2/E:CDK4 Compounds structurallyrelated to BTD (NSC645787) Formula 5.0 >100 >20 >100 >20 >100 >20 6Formula 1.4 >100 >71.5 >100 >71.4 >100 >71.4 6 Formula1.7 >100 >58.8 >100 >58.8 >100 >58.8 7

An IC₅₀ of 10 μM is generally considered effective for these compounds,but effectiveness should be considered in the light of specificity forCDK4.

EXAMPLES

The following examples are provided to illustrate certain features ofthe invention and are not meant to limit the invention to any particularembodiment.

Example 1

This example describes in detail how the compounds of the invention wereidentified and tested to determine their specific inhibitory activityagainst cyclin dependent kinases. Essentially, the methods of thisexample include three stages: (1) determining which cell lines containp16 alterations, (2) determining which drugs are most active against p16altered cells, and (3) determining the CDK4 kinase inhibitory activityof selected, screened compounds.

Methods

Cell lines, compounds, and in vitro sensitivity testing. Exponentiallygrowing cultures of the nine non-small cell lung, eight melanoma, eightrenal, eight breast, seven colon, six brain, six leukemia, six ovarian,and two prostate cancer cell lines from the NCI drug screen panel wereused. Compounds were obtained from the Drug Synthesis and ChemistryBranch, National Cancer Institute. In vitro antitum or activity ofcompounds was determined using a sulforhodamine-B assay in the 60 humancancer cell lines of the NCI drug screen panel.

Polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and DNA sequence analysis of p16. Approximately 1.5×10⁵ tumorcells were washed with PBS, lysed in 100 μl proteinase K solution [200mg/ml, 50 mM Tris-HCl (pH8.5), 1 mM EDTA(pH8.0), and 0.5% Tween, 20],and incubated at 50° C. for 4 h. One microliter of this lysate was usedas template in a 10 μl PCR for each of seven oligonucleotide primerpairs which span the coding region and splice junctions of exons 1 and 2of p16 twice. SmaI-digested (for primer pair 2D) or undigested PCRproducts were subjected to SSCP. The presence of bands with an abnormalmigration pattern was confirmed by repeating PCR-SSCP at least onceprior to extraction of the band, cloning into pT7Blue(R) T-vector(Novagen, Madison, Wis.), and DNA sequence analysis by the dideoxy chaintermination method using Sequenase™ (US Biochemical, Cleveland, Ohio).The presence of intact genomic DNA was confirmed by amplification of a536-bp fragment of the β-globin gene. The p16 sequence published byOkamoto et al. (GenBank accession number L27211) was used as referencefor DNA and amino acid numbering.

Reverse Transcription (RT)-PCR and Southern blot hybridization analysesof p16. Total RNA was isolated from 1×10⁶ cells of each cell line usingan RNA isolation kit (5′ prime 3′ prime,Inc., Boulder, Colo.), RT-PCRwas performed for the p16 gene as previously described. PCR productswere separated by agarose gel electrophoresis, transferred to a nylonmembrane, and hybridized with a 388-bp p16 exon 1 genomic fragmentdefined by oligonucleotides 2F and 1108R. Expression of theglyceraldehyde-3-phosphate (GAPDH) gene was examined to assure thepresence of intact mRNA in each sample by addition of a gene-specificoligonucleotide, G3PD-2R (5′-GATACATGACAAGGTGCGGC-3′) to the reversetranscriptase reaction followed by 40 cycles of PCR (30 sec at 94° C.,30 sec at 55° C., and 1 min at 72° C. using oligonucleotides, G3PD-1F(5′TCGTGGAAGGACTCATGACC-3′) and G3PD-1R (5′ ACATGGCAACTGTGAGGAGG-3′).

Immunoblot analysis. Cells (1×10⁷) were washed with PBS, resuspended in0.4 ml of lysis buffer [50 mM Tris-HCl (pH7.4), 250 mM NaCl, 5 mM EDTA,0.1% Nonidet P40, 50 mM NaF, and 1 mM PMSF], and centrifuged at 14,000rpm for 20 min at 4° C. The protein concentration of the supernatant wasdetermined using the Bio-Rad protein assay reagent (Bio-Rad, Hercules,Calif.). Fifty micrograms of total protein were mixed with an equalvolume of 2×sample buffer [125 mM Tris-HCl (pH 6.8), 20% glycerol, 4%(w/v) SDS, 0.005% bromophenol blue, and 5% 2-mercaptoethanol], loaded ona 14% Tris-glycine gel, and subjected to electrophoresis at 125 V for 90min in 1×running buffer (25 mM Tris-base, 192 mM glycine, and 0.1% SDS).The separated proteins were transferred to a nitrocellulose membrane at25 V for 2 h in transfer buffer (12 mM Tris-base, and 96 mM glycine, 20%methanol). After 30 min incubation at room temperature in blockingsolution (1×PBS, 5% powdered dry milk, and 1% BSA), the membrane wasincubated at 4° C. with 1:1000 dilution of polyclonal anti-human p16antiserum (PharMingen, San Diego, Calif.) overnight, rinsed 5 times withPBS, incubated with a mixture of 40 μl ¹²⁵I-Protein A (>30 mCi/mg) in 20ml blocking solution at 4° C. for one hour, washed again with PBS, airdried for 15 min, and subjected to autoradiography.

COMPARE analysis. The COMPARE algorithm was performed. For theidentification of agents with differential activity, “G150” values of 0and 1 were used for p16-normal and for p16-altered cell lines,respectively. p16-altered cell lines were those with biallelic deletion,intragenic mutation, or transcriptional suppression of p16 andp16-normal cell lines were those without these abnormalities. Pearsoncorrelation coefficients were calculated by the SAS procedure PROC CORR(SAS Institute Inc., Cary, N.C.).

GST fusion proteins. Full length p16 cDNA from cell lines containingintragenic mutations (NCI-H69, MDA-MB-435, UACC-257, and DU-145) wereproduced by RT-PCR using oligonucleotides MK52(5′CGTGAATTCAAGCTTCCTCTCTGGTTCTTTCAATCGGG-3′) and MK68(5′GATGGGATCCCGGCGGCGGGGAGCAGC-3′), cloned into pGEX-5X-1 plasmid(Pharmacia Biotech, Piscataway, N.J.) and sequenced. A GST-Rb fusionplasmid encoding the larger “pocket” domain of Rb was used andGST-fusion proteins were expressed in E. coli (DH5α) and purified usingglutathione sepharose (Pharmacia Biotech, Piscataway, N.J.) according tomanufacturers recommendations.

In vitro kinase assay. Seventy-two hours after infection of 1×10⁷ Sf9cells with baculovirus containing a human CDK gene and/or a cyclin gene,cells were lysed in 250 μl of lysis buffer [50 mM HEPES (pH 7.5), 10 mMMgCl₂, 1 mM DTT, 5 ig/ml of aprotinin, 5 μg/ml of leupeptin, 0.1 mM NaF,0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM sodiumorthovanadate], centrifuged, and lysates stored at −70° C. Fivemicroliters of CDK:cyclin lysate were mixed with test compounds in 40 μlof kinase buffer (200 mM Tris-HCl, pH 8.0, 100 mM MgCl₂, 10 mM EGTA) andincubated at 30° C. for 30 min. About 400 ng of purified GST-Rb fusionprotein and 5 μCi of γ-[³²P]ATP were added to the mixture and incubatedat 30° C. for 15 min. Reactions were stopped by the addition of 250 μlof IP buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl, 0.5% NP-40) and 15 μlglutathione sepharose. After one hour incubation at 4° C., sepharosebeads were washed four times with IP buffer, mixed with 18 μl of2×sample buffer and electrophoresed on an 8% Tris-glycine gel (Novex,San Diego, Calif.) at 125 V for 90 min. Equal recovery of GST-Rb fusionprotein was confirmed by Coomassie blue staining prior toautoradiography.

CDK4 binding assay. Sf9 cells (1×10⁷) were co-infected with baculoviruscontaining a cloned human CDK4 gene and/or a cyclin D1 gene in 12.5 mlof Grace's insect medium (Paragon, Baltimore, Md.) containing 10% FBS.After 40 h, cells were washed and placed in 5 ml of methionine-freemedium containing 200 μCi/ml of [³⁵S]methionine (1000 Ci/mmole) for 4 h,followed by lysis in 250 μl. Cleared cell lysate (10 μl) was incubatedwith 400 ng of wildtype or mutant GST-p16 fusion proteins using the sameconditions as the in vitro kinase assay. After a 30 min incubation,GST-p16 fusion protein was separated using glutathione sepharoseaccording to manufacturer's recommendations, and electrophoresed on a14% Tris-glycine gel (Novex, San Diego, Calif.). The gel was stainedusing Coomassie blue, dried, and autoradiography was performed. Equalrecovery of GST-p16 fusion protein was confirmed by Coomassie bluestaining. To test the effect of compounds on p16 binding to CDK4, 100 μMof each compound was incubated with CDK4:cyclin D1 lysate for 30 minprior to adding GST-p16 fusion protein.

Results

Characterization of the p16 status of the cell lines of the NCI drugscreen panel. To detect genetic alternations of p16 in the 60 cell linesof the NCI drug screen panel, polymerase chain reaction-single strandconformation polymorphism (PCR-SSCP) analysis was performed for exons 1and 2 of the p16 gene using genomic DNA. Exon 3, which encodes only fouramino acids, was not examined as mutations limited to exon 3 have notbeen described. Among the 60 cell lines, 29 cell lines were found tolack amplifiable genomic sequences of one or both exons, indicative of abiallelic deletion involving p16. The presence of amplifiable genomicDNA in each sample was confirmed by amplification of a 536 bp fragmentof the β-globin gene. Eight of the 60 cell lines contained areproducible abnormally migrating SSCP band. DNA sequence analysis ofclones of these eight abnormally migrating SSCP fragments revealedalteration of the primary sequence in each. One of these eight celllines, HL-60, had two sites of sequence variation in exon 2 of p16, oneof which was a common polymorphism at codon 148 (A148T). Thispolymorphism, which does not affect p16 function, was also present inthe colon carcinoma cell line, KM12. Additional sequence variants notknown to be polymorphisms were observed in seven (12%) of the 60 celllines. HL-60 contained a nonsense mutation at codon 80 and HCT-116contained a one bp insertion at codon 22-23, which results in aframeshift at codon 22 and termination after codon 42. Both of thesemutations were reasoned to cause loss of p16 function. Three cell lines(MDA-MB-435, MDA-N, and M14) contained the same splice site mutation [Tto C substitution at nucleotide 2 of intron 1 (I1+2^(T-C))], and 2 celllines (UACC-257 and DU-145) had distinct missense mutations. The splicesite mutation resulted in aberrant splicing creating a shortened mRNAthat had deletion of codons 28 to 50. The functional effect of thesplice site and missense mutations was assessed by measuring the bindingof GST-p16 fusion proteins to CDK4. Binding of mutant GST-p16 fusionproteins (I1+2^(T-C), D84Y, and P81L) to CDK4 was 3.2%, 4.9%, and 34% ofthe binding ability of normal p16, respectively (p<0.0001 for eachcomparison, 2-tailed Student t-test). Thus, 36 of 60 (60%) cell lines ofthe NCI drug screen panel contained a genetic alteration (homozygousdeletion or intragenic mutation) of p16 that disrupted the function ofp16^(INK4A).

To detect non-genetic alterations associated with loss of p16 function,p16 mRNA and protein expression were examined. Using RT-PCR andsubsequent Southern blot hybridization analyses, p16 mRNA expression wasundetectable in 41 of 60 (68%) cell lines examined, including 11 of 24(46%) without detectable genetic alteration. The amplified p16 cDNAs intwo cell lines (MDA-MB435 and MDA-N) were smaller than expected,consistent with altered mRNA splicing as a result of the I1+2^(T-C)mutation. p16 mRNA was not detected in the third cell line (M14) withthis splice site mutation. A protein of 16 kd was detected in 17 of the60 (28%) cell lines by Western blot analysis using p16 polyclonalantiserum. The cell line with a nonsense mutation (HL60) expressed p16mRNA but not p16 protein. The two cell lines with missense mutations(UACC-257 and DU-145) expressed both mRNA and protein. In UACC-257, aprotein smaller than 16 kd was detected, perhaps the result of alteredsusceptibility to proteolysis of p16^(P81L). A protein of 16 kd wasdetected in two cell lines with the splice site mutation (MDA-MB435 andMDA-N) but was absent in the third cell line with the I1+2^(T-C)mutation, M14. In each cell line, absent or altered p16 protein could beattributed to mutation or transcriptional suppression. In total, 47 ofthe 60 (78%) cell lines of the NCI drug screen panel had an alterationof p16.

Comparison of p16 status with growth inhibitory activity. To identifycompounds more active against p16-altered cells than p16-normal cells,the p16 status of the 60 cell lines was matched to the growth inhibitory(GI₅₀) activity of the compounds of the NCI drug screen program andranked according to Pearson correlation coefficients using the COMPAREalgorithm. The growth inhibitory activity of cephalostatin 1, adisteroidal alkaloid extracted from the marine worm, Cephalodiscusgilchristi, correlated best with p16 status (r=0.599). The growthinhibitory activity of five related compounds [cephalostatins 7, 9, 8, 4and 3 were also positively correlated with p16 status (r=0.504, 0.493,0.491, 0.461, and 0.458, respectively). Bryostatin 1, a protein kinase Cactivator isolated from the marine bryozoan, Bugula neritina, had acorrelation coefficient of 0.469.

Aliquots of 26 of the 40 compounds with the highest Pearson correlationrankings were available for further in vitro analysis. These compoundswere assessed for CDK4:cylin D kinase inhibitory activity usingbaculovirus-expressed human CDK4 and cyclin D1, and a GST-Rb fusionprotein as substrate. Six of the 26 compounds examined inhibitedphosphorylation of Rb protein by CDK4:cyclin D1 complex with IC₅₀ valuesranging from 6.8 to more than 100 μM. No inhibition of GST-Rbphosphorylation by CDK4:cyclin D1 was observed in the presence of theother 20 compounds at concentrations up to 100 μM. The most potentinhibitor was 3-amino-9-thio(10H)-acridone (3-ATA; Formula 3) with anIC₅₀ of 6.8 μM, a value similar to the mean GI₅₀ (30 μM) observed forthis compound in the 2 day growth assay of the NCI drug screen.Cephalostatin 1, which has potent antitum or activity in vitro (ED₅₀ ₁₀⁻⁷ to 10⁻⁹ μg/ml), had an IC₅₀ for CDK4:cyclin D1 of 20 μM andbryostatin 1 had no inhibitory activity at the highest concentrationexamined (100 μM).

Characterization of 3-ATA. To examine the specificity of 3-ATAinhibitory activity for CDK4:cyclin D1 kinase, we performed in vitrokinase assays using baculovirus-expressed human CDC2:cyclin A,CDK2:cyclin A, and CDK2:cyclin E complexes. 3-ATA was a less potentinhibitor of CDC2 and CDK2 kinase activities with IC₅₀ values at leastnine-fold higher compared to the IC₅₀ for CDK4. The addition of 100 μM3-ATA decreased the binding of CDK4 to normal p16 by 70% in the p16-CDK4binding assay (p<0.0001, 2-tailed Student t-test), suggesting that 3-ATAmay be acting by a mechanism similar to p16. In the CDK4 kinase assay,the addition of exogenous ATP (0 to 600 μM) did not alter the inhibitoryactivity of 3-ATA, suggesting that 3-ATA was not competing with ATP.Thus, 3-ATA appears to inhibit cyclin-dependent kinase activity by amechanism distinct from that of the flavone L86827 and butyrolactone I,which are known to compete with ATP.

Identification of CDK4-specific inhibitors. To identify compounds in theNCI drug screen that may have a similar mechanism of action as 3-ATA,the pattern of growth inhibitory activity (GI₅₀) of 3-ATA with the GI₅₀of all previously tested compounds as compared. Six compounds notpreviously examined for CDK4 kinase inhibitory activity had similarpatterns of growth inhibitory activity with correlation coefficientsgreater than 0.6. Among these six, two benzothiadiazine (BTD) compounds(Compound 6) and NSC 645788) inhibited CDK4:cyclin D1 kinase activity invitro with IC₅₀'s (5.0 and 17 μM, respectively) similar to the IC₅₀ of3-ATA (6.8 μM).

An additional 45 compounds with structural similarity to 3-ATA and(Compound 6) were available for analysis. Nineteen of these compoundsinhibited CDK4 kinase activity with IC₅₀'s ranging from 1.1 to more than100 μM. Four compounds, 2 structurally related to 3-ATA (Compound 4) andNSC 645153), and 2, Compound 7 and Compound 8, were more potent CDK4kinase inhibitors than the parent compounds. Compound 4, Compound 7, andCompound 8 also had no CDC2 or CDK2 kinase inhibitory activity atconcentrations up to 100 μM. However, two of these compounds, Compound 4and Compound 7, did not inhibit p16^(INK4A) binding to CDK4, suggestingthat their mechanism of inhibition of CDK4 kinase activity is distinctfrom 3-ATA.

Example 2

This example describes a method for treating cancer using the compoundsof the invention. Thioacridones or benzothiadiazines satisfying Formulas1 and 2 above are obtained that specifically inhibit CDK4:cyclin kinasesuch that these compounds have an IC₅₀ for CDK4 that is smaller thantheir IC₅₀ for CDC2 or CDK2. These compounds are administeredintravenously or orally to humans at a dose of between 1 μg and 10grams, preferable between 1 mg and 900 mg per m² of body surface of thepatient. The compounds also can be mixed with at least one additiveselected from the group consisting of carriers, diluents, excipients,diagnostics, direct compression buffers, buffers, stabilizers, fillers,disintegrates, flavors, colors, and mixtures thereof to formpharmaceutical compositions. The compositions are administeredintravenously or orally to humans at a dose of between 1 μg and 10grams, preferable between 1 mg and 900 mg per m² of body surface of thepatient.

Cell Line Data

Compounds of the present invention have been subjected to the drugscreening procedure employed by the National Cancer Institute for thescreening of drugs having possible anticancer utility. The screeningprocedure uses a diverse, disease-oriented panel consisting of differenthuman tumor cell lines organized into disease-specific subpanels. Thecompounds of the present invention were tested over a range ofconcentrations for cytotoxic or growth-inhibitory effects against celllines comprising the panel. The subpanels represented diversehistologies (leukemias, melanomas, and tumors of the lung, colon,kidney, breast, ovary, and brain). The tests produced individualdose-responses, one for each cell line (i.e., one for each example), andthe data are disclosed in dose-response curves, e.g., FIGS. 1(A)-1(I).The data provided by these dose response curves are summarized using amean-graph format, e.g., FIG. 2.

To produce data for the mean-graph format, a compound concentration thatproduced a target level response was calculated for each cell line.Three different response parameters were evaluated. The first responseparameter was the growth inhibition (“GI₅₀”). GI₅₀ is the concentrationof compounds made according to the present invention that produced anapparent 50% decrease in the number of tumor cells relative to theappropriate control (not exposed to the compounds of the presentinvention) at the end of the incubation period.

The second response parameter was the total growth inhibition (“TGI”).TGI is the concentration at which the number of tumor cells remaining atthe end of the incubation period substantially equal the number of tumorcells existing at the start of the incubation period.

The third response parameter was the lethal concentration (“LC₅₀”). LC₅₀is the concentration of compounds made according to the presentinvention that caused an apparent 50 percent reduction in the number oftumor cells relative to the appropriate control (not exposed to thecompounds of the present invention) at the start of the incubationperiod.

In a typical GI₅₀ mean graph the relative position of the verticalreference line along the horizontal concentration axis was obtained byaveraging the negative log₁₀GI₅₀ values for all the cell lines testedagainst the compound. Horizontal bars were then plotted for theindividual negative log₅₀GI₅₀ values of each cell line relative to thevertical reference line. The GI₅₀ graph thus provides a characteristicfingerprint for the compound, displaying the individual cell lines thatare proportionately more sensitive than average (bars extending to theright of the reference line) or proportionately less sensitive thanaverage (bars extending to the left of the reference line). The lengthof a bar is proportional to the difference between the log₁₀GI₅₀ valueobtained with the particular cell line and the mean (represented by thevertical reference line).

The data obtained using the cell line procedures referred to above areprovided by FIGS. 1-12. This data shows that the compounds of thepresent invention inhibit the growth of living cells.

What is claimed is:
 1. A method for treating a mammalian subject havingleukemia, lung cancer, colon cancer, central nervous system cancer,melanoma, ovarian cancer, renal cancer, prostate cancer and/or breastcancer, comprising: providing at least one compound selected from thegroup consisting of compounds having Formula 2

where R and R₁ are carbon or nitrogen, and with R₁=carbon R₁ is bondedto N₁ by a double bond, R is nitrogen, X is hydrogen or halogen, and R₂is selected from the group consisting of alkyl and aryl amino, thecompound having an IC₅₀ for CDK4 of less than about 10 μM, and having anIC₅₀ for CDC2 of more than about 60 μM and having an IC₅₀ for CDK2/A ofmore than about 100 μM, and having an IC₅₀ for CDK2/E of more than about80 μM; and administering an effective amount of the compound to themammalian subject having the cancer.
 2. A method for inhibiting thegrowth of living cancer cells in a mammalian subject having leukemia,lung cancer, colon cancer, central nervous system cancer, melanoma,ovarian cancer, renal cancer, prostate cancer and/or breast cancer,comprising: providing at least one compound selected from the groupconsisting of compounds having Formula 2

where R and R₁ are carbon or nitrogen, and with R₁=carbon R₁ is bondedto N₁ by a double bond and R is nitrogen, X is hydrogen or halogen, andR₂ is selected from the group consisting of alkyl and aryl amino, thecompound having an IC₅₀ ratio for CDC2:CDK4 of more than 8.5, and havingan IC₅₀ ratio for CDK2/A:CDK4 of more than about 14, and having an IC₅₀ratio for CDC2/E:CDK4 of more than about 11.5; and contacting the livingcancer cells of the mammalian subject in vivo, ex vivo or both with anamount of the compound effective to inhibit the growth of the cells. 3.The method according to claim 2 where providing a compound comprisesproviding a composition comprising the compound and additives selectedfrom the group consisting of carriers, diluents, excipients,diagnostics, direct compression buffers, buffers, stabilizers, fillers,disintegrates, flavors, colors, and mixtures thereof.
 4. The methodaccording to claim 2 where, with respect to Formula 2, R is nitrogen. 5.The method according to claim 4 where R₂ is alkyl.
 6. The methodaccording to claim 4 where R₂ is selected from the group consisting ofmethyl and ethyl.
 7. The method according to claim 4 where X is halogen.8. The method according to claim 4 where X is chlorine.
 9. The methodaccording to claim 2 where the compound is


10. The method according to claim 2 where the compound is


11. A method for treating a mammalian subject having leukemia, lungcancer, colon cancer, central nervous system cancer, melanoma, ovariancancer, renal cancer, prostate cancer and/or breast cancer, comprising:providing a compound having a formula

where R and R₁ are carbon or nitrogen, and with R₁=carbon R₁ is bondedto N₁ by a double bond and R is nitrogen, X is hydrogen or halogen, andR₂ is selected from the group consisting of alkyl and aryl amino; andcontacting living cancer cells of the mammalian subject having thecancer in vivo, ex vivo or both with an effective amount of thecompound.
 12. A method for treating a mammalian subject having leukemia,lung cancer, colon cancer, central nervous system cancer, melanoma,ovarian cancer, renal cancer, prostate cancer and/or breast cancer,comprising: providing a compound having a formula

 and contacting living cancer cells of the mammalian subject in vivo, exvivo or both with an effective amount of the compound.